Full text of "USPTO Patents Application 10687850"

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(19)

Europafsches Patentamt
European Patent Office
Office europeen des brevets

(12)

01) EP 0 537 761 B1

EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention
of the grant of the patent:
27.08.1997 Bulletin 1997/35

(21) Application number: 92117711.9

(22) Date of filing: 16.10.1992

(51) Intel* C12M 1/40, C12Q 1/26,
G01N 27/327

(54) A biosensor and a method for measuring a concentration of a substrate in a sample

Biosensor und Verfahren zur Messung einer Konzentration eines Substrats in eine Probe
Biocapteur et methode pour la mesure d'une concentration d'un substrat dans un echantillon

CO
lO

Q.
LU

(84) Designated Contracting States:
DE FR GB

(30) Priority: 18.10.1991 JP 270839/91

21.10.1991 JP 272293/91

09.04.1992 JP 88507/92

(43) Date of publication of application:
21.04.1993 Bulletin 1993/16

(60) Divisional application: 96108449.8

(73) Proprietor: MATSUSHITA ELECTRIC INDUSTRIAL
CO., LTD.

Kadoma-shi, Osaka 571 (JP)

(72) Inventors:

• Yoshioka, Toshihiko
Osaka-shi, Osaka (JP)

• Nankai, Shiro
Hirakata-shi, Osaka (JP)

(74) Representative: Marx, Lothar, Dr.

Patentanwalte Schwabe, Sandmair, Marx
Stuntzstrasse 16
81677 Munchen (DE)

EP-A- 0 359 831

(56) References cited:
EP-A- 0127 958
EP-A- 0 502 504

Remarks:

Divisional application 96108449.8 filed on 28/05/96.

Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art.
99(1) European Patent Convention).

Printed by Jo we. 75001 PAWS (FR)

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention:

The present invention relates to a biosensor that
can easily quantify a specific component in a sample
liquid with speed and accuracy, and a method for meas-
uring a concentration of a substrate {a specific compo-
nent) in a sample by using the biosensor. More particu-
larly, it relates to a biosensor that can quantify a specific
component in a sample liquid by reacting the specific
component in the sample liquid to an oxidoreductase
that specifically reacts to the component and then by
quantifying the change of concentration of a material
that has changed through the reaction after a predeter-
mined period of time, and to a method for measuring the
concentration of a substrate in a sample by using the
biosensor.

2. Description of the Prior Art:

Various types of biosensors utilizing specific catal-
yses of enzyme have been recently developed. A sac-
charide biosensor will be described as an example of
such biosensors as follows:

The optical rotation method, the colorimetric meth-
od, the reductimetry method and other methods using
different kinds of chromatographies have been devel-
oped as methods for quantitative analysis of saccha-
rides. However, none of these methods can provide high
accuracy due to the relatively low specificity against sac-
charides. Additionally, the optical rotation method is
easy tooperate but is largely influenced by the operating
temperature. Therefore, it is not appropriate for common
use at home and the like.

The saccharides contained in fruit are generally as-
sessed as saccharine degrees. A refractometer of the
light refraction system is often used for quantifying the
saccharine degree. This refractometer functions by uti-
lizing change of the light refractive index caused by liq-
uid concentration. Therefore, the refractometer of the
light refraction system is influenced by all the compo-
nents dissolved in the sample liquid, for example, by or-
ganic acid such as citric acid or maleic acid that is con-
tained in fruit juice in large amounts when a saccharide
in the fruit is quantified. Thus, accurate quantification by
this refractometer is impossible.

A glucose sensor will now be described as an ex-
ample of a biosensor used in a clinical field.

A conventional method for quantifying glucose con-
tained in blood is to centrifuge blood taken from a patient
and then to measure the thus obtained blood plasma.
This method requires a lot of time as well as labor.
Therefore, a sensor that can directly measure glucose
in blood obtained from a patient is desired.

A sensor similar to a test paper for urinalysis has

been developed as a simple glucose sensor. This glu-
cose sensor comprises a support in a stick shape and
a holder fixed to the support. The holder includes an en-
zyme reacting only to glucose and a dye, the color of

5 which is changed by reacting with a production of the
enzyme reaction. Blood is dropped onto the support of
the glucose sensor and the change of the color of the
dye after a predetermined period of time of the dropping
is visually observed or optically measured, whereby the

10 content of glucose in the blood can be measured. How-
ever, the quantifying method using this glucose sensor
has low accuracy due to interference by the colored ma-
terials in the blood.

Japanese Laid-Open Patent Publication No.

1* 1-291153 (EP-A-0359831) discloses the following glu-
cose sensor with high accuracy as a method for quan-
tifying a specific component in a sample liquid from a
living body such as blood without diluting or stirring the
sample liquid:

20 The biosensor comprises a base 42, and a spacer
3 and a cover 4 that are laminated integrally onto the
base 42 as is shown in Figures 13 and 14.

The base 42 comprises an electrical insulating sub-
strate 1 , an electrode system 43 formed on the substrate

25 1 by screen printing, etc. and a reaction layer 44 provid-
ed on the electrode system 43. The electrode system
43 includes a working electrode 45 and a counter elec-
trode 46 that are electrically insulated from each other
by an insulating layer 47. The working electrode 45 and

30 the counter electrode 46 are connected to leads 1 2 and
13 formed on the substrate 1, respectively.

The reaction layer 44 includes a hydrophilic poly-
mer, an oxidoreductase and electron acceptors and cov-
ers the working electrode 45 and the counter electrode

35 46.

As is shown in Figure 13, the spacer 3 is in a U-
shape and has a groove 17. When the spacer 3 and the
cover 4 are laminated on the base 42, a passage 18
through which a sample liquid passes is formed be-
40 tween the base 42 and the cover 4 as is shown in Figure
14. One end of the passage 18 is open at one end of
the base 42 and the opening serves as a sample supply
port 23. The other end of the passage 1 8 is open on the
cover 4 and the opening serves as an air port 24.
^5 The operation of the glucose sensor with the above-
mentioned structure is as follows: A sample liquid sup-
plied through the sample supply port 23 reaches the re-
action layer 44 through the passage 1 8 and the oxidore-
ductase and the electron acceptors contained in the re-
50 action layer 44 are dissolved in the sample liquid. Thus,
while an enzyme reaction is proceeded between a sub-
strate in the sample liquid and the oxidoreductase, the
electron acceptors are reduced. After finishing the en-
zyme reaction, the reduced electron acceptors are elec-
ts trochemically oxidized. A value of an oxidation current
obtained at this point provides a concentration of the
substrate in the sample liquid.

However, the conventional biosensor has the fol-

EP 0 537 761 B1

lowing disadvantages:

The sample liquid may include reductive materials
that can reduce the electron acceptors other than the
substrate to be measured. Moreover, viscosity, etc. ot
the sample liquid to be measured vary.

Accordingly, in measuring a concentration of the
substrate in the sample liquid including the substrate
and another reductive material that can reduce the elec-
tron acceptors, the response values of the sensor are
inconstant, and therefore, the reductive material should
be eliminated before the measurement. Such a pretreat-
ment results in increasing the number of steps in meas-
uring the concentration of a substrate in a sample liquid.

Moreover, the sensor response also depends upon
a measuring time. For example, an accurate concentra-
tion can not be obtained when the oxidation current is
measured before completely finishing the reaction.

Furthermore, time required for the sample liquid to
reach the reaction layer and the rate of the reaction of
the substrate to the enzyme depends upon the viscosity
of the sample liquid. Therefore, the inconstant viscosi-
ties result in inconstant sensor responses.

SUMMARY OF THE INVENTION

The biosensor of this invention comprises an elec-
trical insulating substrate, a main electrode system
formed on the substrate and having a working electrode
and a counter electrode, a reaction layer provided in
contact with or in the vicinity of the main electrode sys-
tem and containing an oxidoreductase, and a sub elec-
trode system as a reference provided with an interval
from the main electrode system and having a working
electrode and a counter electrode.

In another aspect of the present invention, the bio-
sensor comprises an electrical insulating substrate, a
main electrode system formed on the substrate and hav-
ing a working electrode and a counter electrode, a re-
action layer provided on the main electrode system and
containing an oxidoreductase and electron acceptors,
and a sub electrode system as a reference provided with
an interval from the main electrode system and the re-
action layer and having a working electrode and a coun-
ter electrode, wherein a substrate contained in a sample
liquid is quantified by reducing the electron acceptors
by electrons generated in a reaction of the substrate
contained in the sample liquid and the oxidoreductase
and then by electrochemically measuring the amount of
the reduced electron acceptors.

Alternatively, the present invention provides a
method for quantifying a substrate contained in a sam-
ple liquid by using a biosensor. The biosensor compris-
es an electrical insulating substrate, a main electrode
system formed on the substrate and having a working
electrode and a counter electrode, a reaction layer pro-
vided in contact with or in the vicinity of the main elec-
trode system and containing an oxidoreductase, and a
sub electrode system as a reference provided on the

substrate with an interval from the main electrode sys-
tem and having a working electrode and a counter elec-
trode. The method comprises the steps of detecting a
presence of the sample liquid on the both the electrode
5 systems by detecting a change of electrical character-
istics between the main electrode system and the sub
electrode system and then applying a voltage to the
main electrode system and a sub electrode system, re-
spectively.

10 Alternatively, the present invention provides a
method for quantifying a substrate contained in a sam-
ple liquid by using a biosensor. The biosensor compris-
es an electrical insulating substrate, a main electrode
system formed on the substrate and having a working

15 electrode and a counter electrode, a reaction layer pro-
vided in contact with or in the vicinity of the main elec-
trode system and containing an oxidoreductase, and a
sub electrode system provided on the substrate with an
interval from the main electrode system and having a

20 working electrode and a counter electrode. The method
comprises the steps of detecting a change of electrical
characteristics between the working electrode and the
counter electrode in the main electrode system and the
sub electrode system, respectively and determining a

25 nature of the sample liquid on the basis of the difference
of time required for detecting the electrical characteris-
tics in the main and sub electrode systems.

Thus, the invention described herein makes possi-
ble the advantages of (1 ) providing a biosensor for quan-
go tifying a specific substrate contained in a sample liquid
easily, rapidly and accurately, (2) providing a biosensor
that conducts an accurate measurement without a p re-
treatment for removing a material in a sample liquid that
disturbs a sensor response, and (3) providing a method

35 for quantifying a substrate contained in a sample liquid
in which constant sensor responses are obtained re-
gardless of viscosity of the sample liquid.

These and other advantages of the present inven-
tion will become apparent to those skilled in the art upon

^o reading and understanding the following detailed de-
scription with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

45 Figure 1 is a plan view of a base of a glucose sensor
according to an example of the present invention.

Figure 2 is an exploded perspective view of the glu-
cose sensor of Figure 1 from which a reaction layer is
removed.

50 Figure 3 is a sectional view of the glucose sensor
of Figure 1.

Figure 4 is a plan view of a base of a glucose sensor
according to another example of the present invention.

Figure 5 is a plan view of a base of a glucose sensor
55 according to still another example of the present inven-
tion.

Figure 6 is an exploded perspective view seen from
one side of a glucose sensor according to still another

EP 0 537 761 B1

example of the present invention.

Figure 7 is an exploded perspective view of the glu-
cose sensor of Figure 6 seen from the other side.

Figure 8 is a plan view of a base of the glucose sen-
sor of Figure 6. 5

Figure 9 is a sectional view of the glucose sensor
of Figure 6.

Figure 10 is a plan view of a base of a glucose sen-
sor according to still another example of the present in-
vention. 10

Figure 11 is a sectional view of a glucose sensor
according to still another example of the present inven-
tion.

Figure 12 is a sectional view of a saccharide sensor
according to still another example of the present inven- is
tion.

Figure 13 is an exploded perspective view of a con-
ventional disposable glucose sensor from which a reac-
tion layer is removed.

Figure 14 is a sectional view of the glucose sensor 20
of Figure 13.

DESCRIPTION OF THE PREFERRED
EMBODIMENTS

Throughout the drawings mentioned in the following
description of the examples, the same element has a
common reference numeral. Part of the description is
omitted as occasion demands.

Example 1

A glucose sensor will now be described as an ex-
ample of a biosensor according to the present invention.

The glucose sensor comprises a base 2, and a 35
spacer 3 and a cover 4 integrally laminated on the base
2 as is shown in Figures 2 and 3.

The base 2 comprises an electrical insulating sub-
strate 1 made from polyethylene terephthalate, an elec-
trode system formed on the substrate 1 by screen print- 40
ing and the like. The electrode system comprises a main
electrode system 1 9 and a sub electrode system 20 pro-
vided on the substrate 1 with an interval therebetween.
The main electrode system 19 and the sub electrode
system 20 include working electrodes 6 and 8 and coun- 45
ter electrodes 7 and 9, respectively. The working elec-
trodes 6 and 8 and the counter electrodes 7 and 9 are
electrically insulated from each other by an insulating
layer 10.

A lead 12 formed on the substrate 1 is electrically so
connected to the working electrode 6 of the main elec-
trode system 19, a lead 1 3 to the counter electrode 7 of
the main electrode system 19, a lead 14 to the working
electrode 8 of the sub electrode system 20, and a lead
1 5 to the counter electrode 9 of the sub electrode system ss
20.

A reaction layer 5 covers the working electrode 6
and the counter electrode 7 of the main electrode sys-

tem 19. The reaction layer 5 includes carboxy methyl
cellulose (hereinafter called the CMC) as a hydrophilic
polymer, glucose oxidase (EC1 . 1 .3.4; hereinafter called
the GOD) as an oxidoreductase, and potassium ferricy-
anide as electron acceptors.

As is shown in Figure 2, the spacer 3 is formed in a
U-shape and has a groove 17 that is open at one end
thereof. When the spacer 3 and the cover 4 are laminat-
ed on the base 2, a passage 18 through which a sample
liquid passes is formed between the base 2 and the cov-
er 4. One end of the passage 18 is open at one end of
the base 2, and the opening serves as a sample supply
port 23. The other end of the passage 18 is open on the
cover 4, and the opening serves as an air port 24. Ac-
cordingly, the reaction layer 5 is provided between the
air port 24 and the sample supply port 23 so as to face
the passage 18.

The glucose sensor was produced as follows: Silver
paste was printed on the substrate 1 by means of screen
printing to form the leads 12, 13, 14 and 15. Then con-
ductive carbon paste including resin binder was printed
on the substrate 1 to form the working electrode 6 of the
main electrode system 19 and the working electrode 8
and the counter electrode 9 of the sub electrode system
20.

The working electrodes 6 and 8 and the counter
electrode 9 were electrically connected to the leads 1 2,
14 and 15, respectively.

Next, insulating paste was printed on the substrate
1 to form the insulating layer 10. The insulating layer 10
covered the peripheral portion of the working electrode
6 so that a predetermined area of the working electrode

6 was exposed. Further, the insulating layer 10 covered
a part of the leads 12, 13, 14 and 15, respectively. The
working electrode 8 and the counter electrode 9 of the
sub electrode system 20 were partly covered by the in-
sulating layer 10 so that a predetermined area of the
working electrode 8 and the counter electrode 9 were
respectively exposed.

Then, conductive carbon paste including resin bind-
er was printed on the insulating layer 10 so as to come
in contact with the lead 13 to form the counter electrode

7 of the main electrode system 19. The base 2 shown
in Figure 1 was produced in this manner.

Next, an aqueous solution including the GOD as the
oxidoreductase, potassium ferricyanide as the electron
acceptors and the 0.5 wt% of CMC as the hydrophilic
polymer was dropped on the working electrode 6 and
the counter electrode 7 of the main electrode system 1 9,
and was dried in a warm-air drier at a temperature ot
50°C for 10 minutes to form the reaction layer 5. The
reaction layer 5 can be easily formed in this manner.

Then, a mixed aqueous solution including potassi-
um ferricyanide and the CMC was dropped onto the
working electrode 8 and the counter electrode 9 of the
sub electrode system 20 and dried to form a reference
layer 25.

After forming the reaction layer 5 and the reference

EP 0 537 761 B1

layer 25 on the substrate 1, the cover 4 and the spacer
3 were laminated to be adhered onto the base 2 as
shown in Figure 2 with dashed lines. Thus, the passage
18 having a comparatively small cross section was
formed which was defined by the groove 17 of the spac-
er 3, the cover 4 and the base 2.

The thus produced glucose sensor was supplied
with 3u.l of a mixed aqueous solution including glucose
and ascorbic acid as a sample liquid through the sample
supply port 23. Before the supply of the sample liquid,
the entire biosensor including the reference layer 25 and
the reaction layer 5 was in a dry condition. The sample
liquid coming in contact with the sample supply port 23
at the tip of the sensor is introduced into the passage
18 by capillarity. Thus, the sample liquid is introduced
into the sub electrode system 20 and the reaction layer
5 by simply allowing the sample liquid to come into con-
tact with the sample supply port 23.

The sample liquid reached the air port 24 through
the sub electrode system 20 due to capillarity, and the
reference layer 25 on the sub electrode system 20 and
the reaction layer 5 on the main electrode system 19
were respectively dissolved in the sample liquid. At the
same time, an impedance between the working elec-
trode 6 of the main electrode system 1 9 and the working
electrode 8 of the sub electrode system 20 was
changed. The impedance change showed a sufficient
supply of the sample liquid to the sensor. Next, a voftage
of +0.5 V on the basis of the voltage at the counter elec-
trode 9 of the sub electrode system 20 was applied to
the working electrode 8. An oxidation current (an anodic
current) value t 0 of five seconds after the application was
measured.

The potassium ferricyanide on the sub electrode
system 20 was reduced by ascorbic acid in the sample
liquid to generate potassium ferrocyanide. The oxida-
tion current value l 0 obtained by the application of the
above-mentioned predetermined voltage was caused
by oxidation of the potassium ferrocyanide. Therefore,
the oxidation current value l 0 was in proportion to the
amount of ascorbic acid contained in the sample liquid.

A voltage of +0.5 V on the basis of the voltage at
the counter electrode 7 of the main electrode system 19
was applied to the working electrode 6 of the main elec-
trode system 19 one minute after detecting the above
described impedance change. An oxidation current val-
ue l 1 of five seconds after the application was measured.

The oxidation current vatue l t is a total value of a
current caused by oxidation of potassium ferrocyanide
generated by reduction by ascorbic acid and one
caused by oxidation of potassium ferrocyanide gener-
ated in oxidizing glucose by the GOD.

When a coefficient for correcting the difference in
the responses in both the electrode systems is taken as
k, a current value represented as Ij-Wq closely corre-
sponded to the glucose concentration in the sample liq-
uid.

Next, responses obtained in the following opera-

tions (1) and (2) were compared, using thirty glucose
sensors.

(1) After supplying the sample liquid to the sensor
5 through the sample supply port; a sufficient supply

of the sample liquid to the reaction layer 5 was de-
tected by detecting the change of the electrical
characteristics between the main electrode system
and the sub electrode system. Then the sensor re-
io sponses were obtained in the above-mentioned
manner.

(2) Instead of the detection of the change in the
electrical characteristics, sufficient supply of the

'5 sample liquid to the reaction layer 5 was confirmed
by visual observation. Then the sensor responses
were obtained by measuring the oxidation currents
value l 0 and Ij in the same manner as above.

20 As a result, a coefficient of variation (a CV value)
showing the dispersion of the responses was 2% in op-
eration (1) and 5% in operation (2).

As mentioned above, the coefficient of the variation
in operation (1) was smaller than that in operation (2).

2S This seems to be because the inconstancy in time from
the measurement of the response current in the sub
electrode system to that in the main electrode system
was made smaller in operation (1).

In this example, the GOD in the reaction layer 5 was

30 not immobilized in the main electrode system 19. How-
ever, since the reaction layer 5 contained the hydrophilic
polymer, the viscosity of the sample liquid was en-
hanced when the reaction layer 5 was dissolved in the
sample liquid. Therefore, dispersion of materials con-

3S tained in the reaction layer 5 was prevented so that the
materials did not move toward the sub electrode system
20 in a short period of time. A more accurate measure-
ment can be effectively attained by immobilizing an en-
zyme such as the GOD in the main electrode system 1 9.

Example 2

Figure 4 is a plan view of a base 2 of a glucose sen-
sor produced as another example of the biosensor ac-
45 cording to the present invention. This glucose sensor
was produced as follows:

Silver paste was printed on an electrical insulating
substrate 1 made from polyethylene terephthalate by
screen printing to form leads 12, 13, 14 and 15. Next,
so conductive carbon paste including resin binder was
printed on the substrate 1 to form a working electrode 6
of a main electrode system 19 and a working electrode
8 of a sub electrode system 20.

The working electrodes 6 and 8 were electrically
55 connected to the leads 12 and 14, respectively.

Insulating paste was then printed on the substrate
1 to form an insulating layer 10. The insulating layer 10
covered peripheral portions of the working electrodes 6

EP 0 537 761 B1

and 8 so that a predetermined area of the working elec-
trodes 6 and 8 was exposed, respectively. Moreover, the
insulating layer 10 covered a part of the leads 12, 13,
14 and 15, respectively.

Next, conductive carbon paste including resin bind-
er was printed on the insulating layer 10 so as to come
in contact with the leads 13 and 15 to form a counter
electrode 7 of the main electrode system 19 and a coun-
ter electrode 9 of the sub electrode system 20. In this
manner, the base 2 shown in Figure 4 was produced.

A mixed aqueous solution including the GOD, po-
tassium ferricyanide and the CMC was dropped on the
working electrode 6 and the counter electrode 7 of the
main electrode system 19 in the same manner as in Ex-
ample 1. A mixed aqueous solution including bovine se-
rum albumin (hereinafter called the BSA), potassium
ferricyanide and the CMC was then dropped on the
working electrode 8 and the counter electrode 9 of the
sub electrode system 20, and dried in a warm-air drier
at a temperature of 50°C for 10 minutes, to form a re-
action layer and a reference layer (not shown) on the
main electrode system 19 and the sub electrode system
20, respectively.

Then a spacer 3 and a cover 4 were laminated in-
tegrally on the base 2 having the reaction layer and the
reference layer in the same manner as in Example 1, to
form the glucose sensor.

Since the reference layer comprising the BSA, po-
tassium ferricyanide and the CMC is formed on the sub
electrode system 20, conditions for diffusion of a reduc-
tive material such as ascorbic acid on the sub electrode
system 20 and the like, are similar to those on the main
electrode system 19.

In the case that proteins such as the GOD and the
BSA exist on the electrode systems, the activity of the
electrodes may be partly degraded by absorption of
such proteins. However, a layer including proteins pro-
vided on both the main and sub electrode systems 19
and 20 as described above can minimize errors in the
oxidation current values detected in each electrode sys-
tem due to the absorption of the proteins. As a result,
the correction between the oxidation current values in
both the electrode systems 19 and 20 can be simplified.

Such an advantage is especially remarkable in a
sensor using carbon as a main electrode material.

Example 3

Next, a fructose sensor as an example of a biosen-
sor will be described.

Figure 5 is a plan view of a base 2 of a fructose
sensor produced as still another example of the biosen-
sor according to the present invention. The fructose sen-
sor was produced as follows:

As is shown in Figure 5, silver paste was printed on
an electrical insulating substrate 1 made from polyeth-
ylene terephthalate by screen printing to form leads 12,
1 3 and 14. Next, conductive carbon paste including res-

in binder was printed on the substrate 1 to form a work-
ing electrode 6 of a main electrode system 19 and a
working electrode 8 of a sub electrode system 20. An
insulating layer 10 was then formed on the substrate 1
5 by using insulating paste. The insulating layer 10 cov-
ered propheral portions of the working electrodes 6 and
8 so that a predetermined area of the working electrodes
6 and 8 was respectively exposed. Further, the insulat-
ing layer 10 covered a part of the leads 12, 13 and 14,

10 respectively.

Next, conductive carbon paste including resin bind-
er was printed on the insulating layer 10 so as to come
in contact with the lead 13 to form a counter electrode
7. Thus the base 2 was produced.

*5 Next, a mixed aqueous solution of fructose dehy-
drogenase (EC 1 . 1 .99. 1 1 ; hereinafter called the FDH) as
an oxidoreductase, potassium ferricyanide as electron
acceptors and the CMC as a hydrophilic polymer in a
phosphate buffer solution (pH = 5) was dropped on the

20 working electrode 6 and the counter electrode 7 of the
main electrode system 19, and dried in a warm-air drier
at a temperature of 40°C for 10 minutes to form a reac-
tion layer (not shown).

A spacer and a cover were laminated integrally on

25 the thus obtained base 2 in the same manner as in Ex-
ample 1 to produce the fructose sensor.

Three jit of a mixed aqueous solution of fructose
and ascorbic acid was supplied to the fructose sensor.
A voltage of +1 V on the basis of the counter electrode

30 7 was applied to the working electrode 8 of the sub elec-
trode system 20 and an oxidation current value l 0 was
measured. Since no oxidoreductase and electron ac-
ceptors existed on the working electrode 8 of the sub
electrode system 20, the current value l 0 was an oxida-

35 tion current value of ascorbic acid contained in the sam-
ple liquid. Moreover, since no hydrophilic polymer and
the like for preventing diffusion of materials existed on
the sub electrode system, an accurate current value l 0
was obtained immediately after the supply of the sample

40 liquid. The current value l 0 was in proportion to the
ascorbic acid concentration.

Moreover, a voltage of +0.5 V on the basis of the
counter electrode 7 was applied to the working electrode
6 of the main electrode system 19 one minute after the

45 supply of the sample liquid to the sensor through the
sample supply port. A current value l t of 5 seconds after
the application was measured. The current value l 1 is a
total value of an oxidation current of potassium ferrocy-
anide generated by a reaction with ascorbic acid and

50 one generated in a reduction of fructose by the FDH.

The amount of ascorbic acid contained in the sam-
ple liquid was quantified by using the current value l 0 . A
fructose concentration in the sample liquid was calcu-
lated from the quantified amount of ascorbic acid and

55 the amount of potassium ferrocyanide quantified with
the current value l v

In the fructose sensor according to this example,
the counter electrode 7 serves as a common counter

EP 0 537 761 B1

electrode to the main and sub electrode systems 1 9 and
20. The production of the sensor can be simplified by
using the counter electrode in common in this manner.
Moreover, the cost for producing the sensor can be re-
duced by using one less lead. In addition, inequality of s
the surfaces of the electrode systems on the substrate
1 can be minimized, thereby preventing the reaction lay-
er from peeling off from the electrode systems. As a re-
sult, a sensor with excellent conservative and stable
properties can be produced, and an accurate sensor re- io
sponse can be obtained by making the movement of the
sample liquids smoother on the electrode systems.

Example 4

A method for measuring a glucose concentration in
whole blood by using the glucose sensor produced in
the same manner as in Example 1 will now be described.

Whole blood was supplied to the glucose sensor
through the sample supply port 23. The entire glucose 20
sensor including the reaction layer 5 was in a dry con-
dition before the supply of the sample liquid. The sample
liquid, that is, the whole blood reached the sub electrode
system 20 first, thereby reducing an impedance be-
tween the working electrode 8 and the counter electrode 25
9 in the sub electrode system 20. The impedance
change was detected through the leads 14 and 15.

Next, the whole blood reached the main electrode
system 19. When the react bn layer 5 on the main elec-
trode system 1 9 was dissolved, an impedance between 30
the working electrode 6 and the counter electrode 7 of
the main electrode system 19 was reduced. The imped-
ance change was detected through the leads 1 2 and 1 3.

When the reaction layer 5 was dissolved in the
whole blood, the glucose in the blood was oxidized by 35
the GOD, and at the same time potassium ferricyanide
was reduced into potassium ferrocyanide. One minute
after the supply of the whole blood to the glucose sensor,
a voltage of +0.5 V on the basis of the counter electrode
7 was applied to the working electrode 6, and an oxida- 40
tion current 5 seconds after the application was meas-
ured. The obtained current value was due to the reduc-
tion of potassium ferrocyanide and was in proportion to
the concentrat bn of glucose, the substrate to be meas-
ured. 45

When the above described oxidation current values
were measured by using the whole blood sample with a
hematocrit of 20% to 60%, a higher hematocrit reduced
the oxidation current value. Moreover, when the differ-
ence of time required for detecting the impedance 50
change between the main electrode system and the sub
electrode system was taken as t, the t increased propor-
tionally to the increase of the hematocrit in using the
whole blood with a hematocrit of 20% to 60%.

When values obtained by correcting the oxidatbn ss
current value with the above factor t were taken as sen-
sor responses, the sensor responses were constant re-
gardless of the hematocrit value in the whole blood sam-

ple and corresponded to the glucose concentration in
the whole blood.

In this example, the sample liquid can be supplied
through the air port 24, using the sample supply port 23
as an air port. In this case, the impedance change is first
detected in the main electrode system, and then in the
sub electrode system. The difference in time t2 required
for detecting the impedance change between the main
electrode system and the sub electrode system does not
necessarily correspond to the above t However, the
same effect as in this example can be attained by stud-
ying the relation between t2 and the hematocrit previ-
ously.

Example 5

A glucose sensor will now be described as an ex-
ample of the biosensor according to the present inven-
tion.

Figure 6 is an exploded perspective view of the glu-
cose sensor seen from one side from which a reaction
layer 50 is removed. Figure 7 is an exploded perspective
view of the glucose sensor seen from the other side from
which a reaction layer 50 is removed. Figure 8 is a plan
view of a base 2 of the glucose sensor produced in this
example. Figure 9 is a sectional view of the glucose sen-
sor produced in this example.

A method for producing the glucose sensor will now
be described.

Silver paste was printed on a substrate 1 made from
polyethylene terephthalate by screen printing to form
leads 12 and 13. Next, conductive carbon paste includ-
ing resin binder was printed on the substrate 1 to form
a working electrode 6 of a main electrode system 19.
The working electrode 6 was electrically connected to
the lead 12. Insulating paste was then printed on the
substrate 1 to form an insulating layer 10. The insulating
layer 10 covered the peripheral portion of the working
electrode 6 so that a predetermined area of the working
electrode 6 was exposed.

Next, conductive carbon paste including resin bind-
er was printed on the insulating layer 10 so as to come
in contact with the lead 13 to form a counter electrode
7 of the main electrode system 19.

On the reverse surface of the insulating substrate 1
on which the above-mentioned electrode pattern was
printed, leads 14 and 15, a sub electrode system 20 (in-
cluding a working electrode 8 and a counter electrode
9) and an insulating layer 16 were formed by printing,
thereby producing the base 2 as shown in Figures 7 and
8.

The structures of the main electrode system 19 and
the sub electrode system 20 were the same, and there-
fore, the areas of the working electrodes 6 and 8 were
also the same.

A mixed aqueous solutbn including the GOD as the
oxidoreductase, potassium ferricyanide as the electron
acceptors and the CMC as the hydrophilic polymer was

EP0 537 761 B1

dropped on the main electrode system 19, and dried in
a warm-air drier at a temperature of 50°C for 10 minutes
to form a reaction layer 50.

Next, spacers 21 and 29 and covers 22 and 26 were
laminated to adhere to the base 2 having the above-de-
scribed reaction layer 50 as shown in Figure 9, to pro-
duce the glucose sensor.

To the thus obtained glucose sensor, 10 uJ of a
mixed aqueous solution of glucose and ascorbic acid as
a sample liquid was supplied through sample supply
ports 23 and 27. The supplied sample liquid immediately
reached air ports 24 and 28 due to capillarity, and the
reaction layer 50 on the main electrode system 19 was
dissolved.

A voltage of +1 V on the basis of the counter elec-
trode 9 of the sub electrode system 20 was then applied
to the working electrode 8, and the current value l 0 was
measured. Since no oxidoreductase and electron ac-
ceptors existed on the sub electrode system 20, the cur-
rent value l 0 was a value of an oxidation current of ascor-
bic acid in the sample liquid. Moreover, since no hy-
drophilic polymer for preventing dispersion of materials
existed on the sub electrode system 20, the current val-
ue l 0 was obtained immediately after the supply of the
sample liquid. The current value l 0 was in proportion to
the concentration ot ascorbic acid.

One minute after the supply of the sample liquid, a
voltage of +0.5 V on the basis of the counter electrode
7 of the main electrode system 19 was applied to the
working electrode 6 of the main electrode system 19,
and the current value I-, after 5 seconds of the applica-
tion was measured. The current value Ij was a total val-
ue of an oxidation current of potassium ferrocyanide
generated by a reaction to ascorbic acid and one gen-
erated in a reduction of glucose by the GOD.

The amount of ascorbic acid in the sample liquid
was quantified from the current value l 0 . A glucose con-
centration in the sample liquid was calculated from the
quantified amount of ascorbic acid and the amount of
potassium ferrocyanide obtained from the current value
"1-

In this example, the main electrode system 19 and
the sub electrode system 20 were provided on different
sides from each other. Accordingly, the materials con-
tained in the reaction layer 50 such as the GOD did not
move onto the sub electrode system 20. As a result,
there was no need to immobilize the GOD, potassium
ferricyanide and the like to the main electrode system
19.

Apart from in the above-mentioned method, the
base 2 can be produced by laminating two insulating
substrates 1 respectively bearing an electrode pattern
on one surface thereof to each other. The structure of
the sub electrode system does not necessarily corre-
spond to that of the main electrode system. For exam-
ple, the structure shown in Figure 10 can be used.

Example 6

Figure 11 is a sectional view of a glucose sensor
produced as yet another example of the present inven-
5 tion.

A base 2 was produced in the same manner as in
Example 5.

A mixed aqueous solution of the GOD, potassium
ferricyanide and the CMC was dropped on a main elec-

io trode system 19 (a working electrode 6 and a counter
electrode 7) of the base 2 and dried to form a reaction
layer 51 in the same manner as in Example 5. Next, a
mixed aqueous solution of potassium ferricyanide and
the CMC was dropped onto a sub electrode system 20

is (a working electrode 8 and a counter electrode 9) and
dried to form a reference layer 25 made from potassium
ferricyanide and the CMC.

A spacer 3 and a cover 4 were integrally laminated
on the base 2 in the same manner as in Example 5 to

20 form the glucose sensor.

To the thus obtained glucose sensor, 10 uJ of a
mixed aqueous solution of glucose and ascorbic acid as
a sample liquid was supplied through sample supply
ports 23 and 27. The sample liquid immediately reached

25 air ports 24 and 28 due to capillarity.

The reference layer 25 was dissolved in the sample
liquid on the sub electrode system 20. Pottasium ferri-
cyanide was reduced by ascorbic acid in the sample liq-
uid. A voltage of +0.5 V on the basis of the counter elec-
ta trode 9 of the sub electrode system 20 was applied to
the working electrode 8 of the sub electrode system 20
ten seconds after the supply of the sample liquid. A cur-
rent value measured 5 seconds after the application was
in proportion to the concentration of ascorbic acid in the

35 sample liquid.

The reaction layer 51 was dissolved in the sample
liquid on the main electrode system 19. Pottasium ferri-
cyanide in the reaction layer 51 was changed into po-
tassium ferrocyanide by two reactions: reduction by

40 ascorbic acid in the sample liquid and reduction in oxi-
dizing glucose in the sample liquid by the GOD.

A voltage of +0.5 V on the basis of the counter elec-
trode 7 of the main electrode system 19 was applied to
the working electrode 6 of the main electrode system 19

45 one minute after the supply of the sample liquid to the
sensor. The current value Ij of 5 seconds after the ap-
plication was measured.

The current value l 1 was a total value of the oxida-
tion current of potassium ferrocyanide generated by a

so reaction with ascorbic acid and one generated by a re-
duction in oxidizing glucose by the GOD.

A concentration of ascorbic acid in the sample liquid
was quantified from the response of the sub electrode
system 20. A glucose concentration in the sample liquid

ss was calculated from the quantified ascorbic acid con-
centration and the amount of potassium ferrocyanide
obtained from the current value \ v

An output current value in the sub electrode system

EP 0 537 761 B1

20 can not be accurately measured when the GOD ex-
ists on the sub electrode system 20. The GOD can be
completely prevented from moving onto the sub elec-
trode system 20 by providing the main electrode system
19 and the sub electrode system 20 on the different sur-
faces of the substrate 1 from each other as in this ex-
ample. As a result, a more accurate measurement can
be attained.

Example 7

A saccharide sensor will now be described as yet
another example of the biosensor according to the
present invention.

Figure 1 2 is a sectional view of the saccharide sen-
sor according to this example. A method for producing
the saccharide sensor is as follows:

A base 2 shown in Figure 12 was produced in the
same manner as in Example 5.

Next, a mixed aqueous solution including the GOD
as the oxidoreductase, potassium ferricyanide as the
electron acceptors and the CMC as the hydrophilic pol-
ymer was dropped on the main electrode system 1 9 and
dried to form a first reaction layer 52.

A mixed aqueous solution including the FDH as the
oxidoreductase, potassium ferricyanide as the electron
acceptors and the CMC as the hydrophilic polymer in a
phosphate buffer (pH = 4.5) was then dropped on the
sub electrode system 20 and dried to form a second re-
action layer 53. A spacer and a cover were laminated
on the base 2 as in Example 5 to produce the saccharide
sensor.

To the thus obtained saccharide sensor, 1 0 uJ of glu-
cose and fructose as a sample liquid was supplied
through sample supply ports 23 and 27. A voltage of
+0.5 V on the basis of the counter electrode 7 and a
voltage of +0.5 V on the basis of the counter electrode
9 were applied to the working electrodes 6 and 8, re-
spectively 2 minutes after the supply of the sample liq-
uid. A current value at each electrode after 5 seconds
was measured. In the main electrode system 1 9 with the
first reaction layer 52, the current value corresponded
to the glucose concentration. In the sub electrode sys-
tem 20 with the second reaction layer 53, the current
value corresponded to the fructose concentration.

When the first and the second reaction layers 52
and 53 were dissolved in the sample liquid, the sub-
strates in the sample liquid were respectively oxidized
with the oxidoreductase specific to each layer. Pottasi-
um ferricyanide was reduced into potassium ferrocya-
nide with an electron transfer in each layer. Next, by the
application of the above predetermined voltages, an ox-
idation current value corresponding to the generated po-
tassium ferrocyanide was obtained. The current value
corresponded to the concentration of the substrate in
the sample liquid.

The sensor response of the above saccharide sen-
sor supplied with fruit juice as a sample liquid was meas-

ured. Glucose and fructose in the fruit juice could be
quantified.

The GOD used in the first reaction layer 52 and the
FDH used in the second reaction layer 53 have different

5 pH conditions for providing the highest enzyme reactiv-
ity from each other. Generally, the most appropriate pH
condition often depends upon a kind of the used en-
zyme. When the first and the second reaction layers 52
and 53 are provided on the same surface of the sub-

io st rate 1 , a buffering component contained in the second
reaction layer 53 moves into the first reaction layer 52
containing the GOD by the dispersion of the sample liq-
uid. Thus, the most appropriate pH condition may not
be obtained. Further, the enzyme may move onto a plu-

15 rality of electrodes by the dispersion of the sample liquid.
Therefore, it is necessary to set a condition for not al-
lowing the enzyme to move by immobilization or the like.
As a result, the structure of the sensor can be limited.
When the reaction layers containing different en-

20 zymes are provided on the different surfaces of the in-
sulating substrate 1 from each other as in this example,
a component in each reaction layer can be prevented
from moving when each reaction layer is dissolved in
the sample liquid. In this way, the pH on each electrode

25 system can be easily settled to be the most appropriate
to the enzyme, and the enzyme can be freely dispersed
in the sample liquid.

In the above described examples, when the cover
and the spacer are made from a transparent material

30 such as a transparent synthetic resin, it is possible to
observe the condition of the reaction layer and the in-
troducing condition of the sample liquid in the passage
from the outside.

In the foregoing examples, in order to supply the

35 sample liquid to the reaction layer more smoothly, a lec-
ithin layer may be formed by developing an organic sol-
vent solution of lecithin through the sample supply port
into the reaction layer and drying thereof.

When the lecithin layer is provided, the sample liq-

40 uid can be supplied even when the passage defined by
the base, the cover and the spacer is not small enough
to cause capillarity.

Since the amount of the sample liquid to be supplied
depends upon the capacity of the passage, there is no

45 need to previously quantify it. In addition, the evapora-
tion of the sample liquid during the measurement can
be minimized, thereby attaining a more accurate meas-
urement.

The sample supply port is not necessarily distin-
50 guishable from the air port. It is possible to supply the
sample liquid through the air port, using the sample sup-
ply port as an air port.

The oxidoreductase such as the GOD in the reac-
tion layer is not especially immobilized to the main elec-
55 trode system. However, since the reaction layer con-
tains the hydrophilic polymer, the dispersion of the ma-
terial is prevented due to increased viscosity of the sam-
ple liquid when the reaction layer is dissolved in the sam-

EP 0 537 761 B1

pie liquid. Therefore, the material making up the reaction
layer does not move onto the sub electrode system in a
short period of time. The enzyme can be effectively im-
mobilized to conduct a more reliable measurement.

When the sample liquid is supplied through the
sample supply port, it is effective to provide the sub elec-
trode system in the vicinity of the sample supply port. In
this way, the sample liquid proceeds toward the air port
through the sample supply port. Therefore, a possibility
for moving the oxidoreductase during the reaction to-
ward the sub electrode system that is provided in the
downstream of the flow of the sample liquid can be re-
duced. However, when the oxidoreductase is immobi-
lized to the main electrode system, this does not cause
any problem.

In Examples 5, 6 and 7, the sub electrode system
has the same electrode pattern as the main electrode
system. However, it does not have to be the same. For
example, the pattern shown in Figure 10 can be used.

There is no need to form all of the reaction layers,
the first reaction layer, the second reaction layer and the
reference layer in contact with the electrode systems as
in the foregoing examples. When the base, the spacer
and the cover are integrated, the reaction layer may be
formed on a reverse surface of the cover and the like so
as to face the passage.

Further, in the above-described examples, a meth-
od for quantifying glucose and fructose is shown. How-
ever, the present invention can be widely used in sys-
tems using an enzyme reaction, as an alcohol sensor,
a lactic acid sensor, a cholesterol sensor and an amino
acid sensor.

Moreover, in the foregoing examples, the GOD and
the FDH are used as the oxidoreductase. However, al-
cohol oxidase, lactase oxidase, lactase dehydroge-
nase, cholesterol oxidase, xanthine oxidase and amino
acid oxidase and the like can be used as well.

The hydrophilic polymer is not limited to the CMC
used in the examples. Other cellulose derivatives such
as hydroxy ethyl cellulose, hydroxy propyl cellulose, me-
thyl cellulose, ethyl cellulose, ethyl hydroxy ethyl cellu-
lose and carboxy methyl ethyl cellulose can be used.
Moreover, the same effect can be attained by using pol-
yvinylpyrrolidone, polyvinyl alcohol, gelatin or its deriv-
atives, acrylic acid or its salts, methacrylic acid or its
salts, starch or its derivatives and maleic anhydride or
its salts.

As electron acceptors, apart from potassium ferri-
cyanide used in the above-mentioned examples, p-ben-
zoquinone, phenazinemethosulfate, methylene blue
and ferrocene derivatives can be used.

Moreover, in the foregoing examples, the oxidore-
ductase and the electron acceptors are dissolved in the
sample liquid. However, they may be immobilized to be
insoluble in the sample liquid.

The above-described electrode system is nof limit-
ed to a two-electrode system having only a working elec-
trode and a counter electrode. A three-electrode sys-

tem, including an additional reference electrode, may be
used, so that more precise values are obtainable.

5 Claims

1. A biosensor comprising:

an electrical insulating substrate (1),
'0 a main electrode system (19) formed on the

substrate (1) and having a working electrode
(6) and a counter electrode (7),
a reaction layer (5;50;51;52) provided in con-
tact with or in the vicinity of the main electrode
*5 system (1 9) and containing an oxidoreductase,

and

a sub electrode system (20) as a reference pro-
vided with an interval from the main electrode
system (1 9) and having a working electrode (8)
20 and a counter electrode (9).

2. A biosensor according to claim 1 , wherein the reac-
tion layer (5;50;51 ;52) further contains electron ac-
ceptors and a hydrophilic polymer.

3. A biosensor according to claim 1 or 2, wherein a
reference layer (25; 53) containing electron accep-
tors and a hydrophilic polymer is provided on the
sub electrode system (20).

4. A biosensor according to any of claims 1 to 3,
wherein the oxidoreductase is selected from the
group consisting of fructose dehydrogenase, glu-
cose oxidase, alcohol oxidase, lactase oxidase,

35 lactase dehydrogenase, cholesterol oxidase, xan-
thine oxidase and amino acid oxidase.

5. A biosensor according to claim 2, wherein the hy-
drophilic polymer is selected from the group con-

40 sisting of carboxy methyl cellulose, hydroxy ethyl
cellulose, hydroxy propyl cellulose, methyl cellu-
lose, ethyl cellulose, ethyl hydroxy ethyl cellulose,
carboxy methyl ethyl cellulose, polyvinylpyrro-
lidone, polyvinyl alcohol, gelatin or its derivatives,

45 acrylic acid or its salts, methacrylic acid or its salts,
starch or its derivatives, and maleic anhydride or its
salts.

6. A biosensor according to claim 2, wherein the elec-
50 tron acceptors is selected from the group consisting

of potassium ferricyanide, p-benzoquinone, phena-
zinemethosulfate, methylene blue and ferrocene.

7. A biosensor according to claim 1 , wherein the main
55 electrode system (1 9) and the sub electrode system

(20) are provided on different surfaces of the sub-
strate (1) from each other, and the reaction (50;51;
52) layer is provided on the main electrode system

EP 0 537 761 B1

(19).

8. A biosensor according to claim 1 , wherein the coun-
ter electrode (7) ot the main electrode system (19)

is common to the counter electrode (9) ot the sub s
electrode system (20).

9. A biosensor according to claim 1 , wherein the main
electrode system (19) is made from a material com-
prising carbon as a main component. w

10. A biosensor according to claim 1, wherein the sub
electrode system (20) is made from a material com-
prising carbon as a main component.

1 1 . A biosensor comprising:

an electrical insulating substrate (1),
a main electrode system (19) formed on the
substrate (1) and having a working electrode 20
(6) and a counter electrode (7),
a reaction layer (5;50;51 ;52) provided on the
main electrode system (19) and containing an
oxidoreductase and electron acceptors, and
a sub electrode system (20) as a reference pro- 25
vided with an interval from the main electrode
system (1 9) and the reaction layer (5;50;51 ;52)
and having a working electrode (8) and a coun-
ter electrode (9),

wherein a substrate contained in a sample liq- 30
uid is quantified by reducing the electron accep-
tors by electrons generated in a reaction of the
substrate contained in the sample liquid and the
oxidoreductase and then by electrochemically
measuring the amount of the reduced electron 35
acceptors.

12. A method for quantifying a substrate contained in a
sample liquid by using a biosensor,

the biosensor comprising an electrical insulat-
ing substrate (1), a main electrode system (19)
formed on the substrate (1 ) and having a work-
ing electrode (6) and a counter electrode (7), a
reaction layer (5;50;51 ;52) provided in contact 45
with or in the vicinity of the main electrode sys-
tem (19) and containing an oxidoreductase,
and a sub electrode system (20) as a reference
provided on the substrate (1) with an interval
from the main electrode system (19) and having so
a working electrode (8) and a counter electrode
(9), and

the method comprising the steps of detecting a
presence ot the sample liquid on both the elec-
trode systems (1 9,20) by detecting a change of 55
electrical characteristics between the main
electrode system (1 9) and the sub electrode
system (20) and then applying a voltage to the

main electrode system (19) and the sub elec-
trode system (20), respectively.

13. A method for quantifying a substrate contained in a
sample liquid by using a biosensor,

the biosensor comprising an electrical insulat-
ing substrate (1), a main electrode (19) formed
on the substrate (1 ) and having a working elec-
trode (6) and a counter electrode (7), a reaction
layer (5;50;51 ;52) provided in contact with or in
the vicinity ot the main electrode system (19)
and containing an oxidoreductase, and a sub
electrode system (20) as a reference provided
on the substrate (1) an interval from the main
electrode system (19) and having a working
electrode (8) and a counter electrode (9), and
the method comprising the steps of detecting a
change of electrical characteristics between
the working electrode (6;8) and the counter
electrode (7;9) in the main electrode system
(1 9) and the sub electrode system (20), respec-
tively and determining a nature of the sample
liquid on the basis of the difference of time re-
quired for detecting the electrical characteris-
tics in the main and sub electrode systems.

Patentanspruche

1. Biosensor mit:

einem elektrisch isolierenden Substrat (1),
einem Hauptelektrodensystem (19), das auf
dem Substrat (1) ausgebildet ist und eine Ar-
bertselektrode (6) und eine Gegenelektrode (7)
aufweist,

eine Reaktionsschicht (5; 50; 51; 52), welche
in Kontakt bzw. in Beruhrung mit oder in der
Umgebung bzw. Nahe des Hauptelektrodensy-
stems (19) ausgebildet ist und eine Oxidore-
duktase enthalt, und

einem Unterelektrodensystem (20) als eine Be-
zugs- bzw. Referenzelektrode, welches in ei-
nem Abstand von dem Hauptelektrodensystem
(19) vorgesehen ist und eine Arbeitselektrode
(8) und eine Gegenelektrode (9) aufweist.

2. Biosensor nach Anspruch 1 , wobei die Reaktions-
schicht (5; 50; 51; 52) weiter Elektronenakzeptoren
und ein hydrophiles Polymer enthalt.

3. Biosensor nach Anspruch 1 oder 2, wobei eine Be-
zugs- bzw. Referenzschicht, (25; 53), welche Elek-
tronenakzeptoren und ein hydrophiles Polymer ent-
halt, auf dem Unterelektrodensystem 20 vorgese-
hen ist.

EP 0 537 761 B1

Biosensor nach einemder Anspruche 1 bis 3, wobei
die Oxidoreduktase aus der Gruppe bestehend aus
Fruktose, Dehydrogenase, Glukoseoxidase, Alko-
holoxidase, Laktaseoxidase, Laktasedehydroge-
nase, Cholesteroloxidase bzw. Cholesterinoxidase, 5
Xanthinoxidase und Aminosaureoxidase gewahlt
wird.

Biosensor nach Anspruch 2, wobei das hydrophile
Polymer aus der Gruppe bestehend aus Carboxy- 10
methylcellulose, Hydroxyethylcellulose, Hydroxy-
propylcellulose, Methylcellulose, Ethylcellulose,
Ethylhydroxyethylcellulose, Carboxymethy I ethyl-
cellulose, Polyvinyl pyrrol idon, Polyvinylalkohol,
Gelatine bzw. Gallert oder dessen Derivate, Acryl- is
saure oder dessen Salze, Methacrylsaure oder des-
sen Salze, Starke oder dessen Derivate und Mal-
einsaureanhydrid oder dessen Salze gewahlt wird.

elektrode bzw. Referenzelektrode, welches in
einem Abstand von dem Hauptelektrodensy-
stem (19) und der Reaktionsschicht (5; 50; 51;
52) vorgesehen 1st und eine Arbeltselektrode
(8) und eine Gegen elektrode (9) aufweist,
wobei ein Substrat, das in einer Probenflussig-
keit enthalten ist, quant rtativ bestimmt wird
durch das Reduzieren der Elektronenakzepto-
ren durch Elektronen, welche bei einer Reakti-
on des Substrats, das in der Probenflussigkeit
enthalten ist und der Oxidoreduktase erzeugt
wurden, und anschlieGend durch elektrochemi-
sches Messen der Menge der reduzierten Elek-
tronenakzeptoren.

12. Vertahren zur quantitativen Bestimmung eines
Substrats, das in einer Probenflussigkeit enthalten
ist unter Verwendung eines Biosensors, wobei

6. Biosensor nach Anspruch 2, wobei die Elektronen-
akzeptoren aus der Gruppe bestehend aus Kalium-
ferricyanid, p-Benzochinon, Phenazinmethosulfat,
Methyl enblau und Ferrocen gewahlt werden.

7. Biosensor nach Anspruch 1 , wobei das Hauptelek-
trodensystem (19) und das Unterelektrodensystem
(20) auf verschiedenen Oberflachen des Substrats
(1) voneinander und die Reaktionsschicht (50; 51;
52) auf dem Hauptelektrodensystem (19) vorgese-
hen sind.

8. Biosensor nach Anspruch 1 , wobei die Gegen elek-
trode (7) des Hauptelektrodensystems (19) mit der
Gegenelektrode (9) des Unterelektrodensystems
(20) gerneinsam ist.

9. Biosensor nach Anspruch 1, wobei das Hauptelek-
trodensystem (19) aus einem Material hergestellt
ist, das Kohlenstoff bzw. Kohle a!s einen Hauptbe-
standteil aufweist.

10. Biosensor nach Anspruch 1, wobei das Unterelek-
trodensystem (20) aus einem Material hergestellt
ist, welches Kohlenstoff bzw. Kohle als einen
Hauptbestandteil aufweist.

11. Biosensor mit:

20 der Biosensor enthalt ein elektrisch isotieren-

des Substrat (1), ein Hauptelektrodensystem
(19), das auf dem Substrat (1) ausgebildet ist
und eine Arbeltselektrode (6) und eine Gegen-
elektrode (7) aufweist, eine Reaktionsschicht
25 (5; 50; 51; 52), welche in Verbindung bzw. in

Kontakt mit oder in der Umgebung bzw. Nahe
des Hauptelektrodensystems (19) ausgebildet
ist und eine Oxidoreduktase enthalt, und ein
Unterelektrodensystem (20) als eine Bezugs-
30 elektrode bzw. Referenzelektrode, welches auf

dem Substrat (1) vorgesehen ist innerhalb ei-
nes Abstands von dem Hauptelektrodensy-
stem (19) und eine Arbe its elektrode (8) und ei-
ne Gegenelektrode (9) aufweist, und
35 das Verfahren weist die Schritte auf: Detektie-

ren des Vorhandenseins der Probeflussigkeit
auf beiden Elektrodensystemen (19, 20) durch
Detektieren einer Veranderung der elektri-
schen Eigenschaften zwischen dem Haupt-
40 elektrodensystem (19) und dem Unterelektro-

densystem (20) und dann Anlegen einer Span-
riung an das Hauptelektrodensystem (19) bzw.
das Unterelektrodensystem (20).

45 13. Verfahren zur quantitativen Bestimmung eines
Substrats, das in einer Probenflussigkeit enthalten
ist unter Verwendung eines Biosensors, wobei

der Biosensor enthalt: ein elektrisch isolieren-
des Substrat (1), ein Hauptelektrodensystem
(19), das auf dem Substrat (1) ausgebildet ist
und eine Arbeitselektrode (6) und eine Gegen-
elektrode (7) aufweist, eine Reaktionsschicht
(5; 50; 51; 52), welche in Verbindung bzw. in
Kontakt mit oder in der Umgebung bzw. Nahe
des Hauptelektrodensystems (19) ausgebildet
ist und eine Oxidoreduktase enthalt, und ein
Unterelektrodensystem (20) als eine Bezugs-

einem elektrisch isolierenden Substrat (1),
einem Hauptelektrodensystem (19), das auf so
dem Substrat (1 ) ausgebildet ist und eine Ar-
beitselektrode (6) und eine Gegenelektrode (7)
aufweist,

einer Reaktionsschicht (5; 50; 51 ; 52), welche
auf dem Hauptelektrodensystem (1 9) vorgese- 55
hen ist und eine Oxidoreduktase und Elektro-
nenakzeptoren enthalt, und
einem Unterelektrodensystem (20) als Bezugs-

EP 0 537 761 B1

elektrode bzw. Referenzelektrode, wefches auf
dem Substrat (1) vorgesehen ist innerhalb ei-
nes Abstands von dem Hauptelektrodensy-
stem (19) und eine Arbeitselektrode (8) und ei-
ne Gegenelektrode (9) aufweist, und 5
das Verfahren weist die Schritte auf: Detektie-
ren einer Veranderung der elektrischen Eigen-
schaften zwischen der Arbeitselektrode (6; 8)
und der Gegenelektrode (7; 9) in dem Haupt-
elektrodensystem (19) bzw. dem Unterelektro- 10
densystem (20) und Bestimmen der Natur der
ProbenflOssigkeit auf der Grundlage bzw. Basis
des Zeit-Unterschieds, welcher zum Detektie-
ren der elektrischen Eigenschaften in den
Haupt- und Unterelektrodensystemen benotigt '5
wird.

Revendications

1. Biocapteur comprenant :

un substrat d'isolation electrique (1),
un systeme d'electrodes principales (1 9) forme
sur le substrat (1 ) et comportant une electrode 25
de travail (6) et une contre-electrode (7),
une couche reactionnelle (5; 50; 51 ; 52) fournie
en contact avec ou dans le voisinage du syste-
me d'electrodes principales (19) et contenant
une oxydoreductase, et 30
un systeme de sous-electrodes (20) comme re-
ference fourni avec un intervalle par rapport au
systeme d'electrodes principales (19) et ayant
une electrode de travail (8) et une contre-elec-
trode (9). 35

2. Biocapteur selon la revendication 1 , dans lequel la
couche reactionnelle (5; 50; 51; 52) contient en
outre des accepteurs d'electrons et un polymere hy-
drophile. 40

3. Biocapteur selon la revendication 1 ou 2, dans le-
quel une couche de reference (25; 53) contenant
des accepteurs d'electrons et un polymere hydro-
phile est fournie sur le systeme de sous-electrodes 45
(20).

4. Biocapteur selon Tune quelconque des revendica-
tions 1 a 3, dans lequel I'oxydoreductase est choisie
dans le groupe constitue des : dehydrogenase de so
fructose, oxydase de glucose, oxydase d'alcool,
oxydase de lactase, dehydrogenase de lactase,
oxydase de cholesterol, oxydase de xanthine et
oxydase d'acide amine.

5. Biocapteur selon la revendication 2, dans lequel le
polymere hydrophile est choisi dans le groupe
constitue des : carboxymethy [cellulose, hydroxye-

thylcellulose, hydroxypropylcellulose, methylcellu-
lose, ethylcellulose, ethylhydroxyethylcellulose,
carboxymethylethylcellulose, polyvinylpyrrolidone,
alcool polyvinyl ique, gelatine ou ses derives, acide
acrylique ou ses sels, acide methacrylique ou ses
sels, amidon ou ses derives, et anhydride maleique
ou ses sels.

6. Biocapteur selon la revendication 2, dans lequel les
accepteurs d'electrons sont choisis dans le groupe
constitue des :ferricyanurede potassium, p-benzo-
quinone, phenazinem6thosulfate, bleu de methyle-
ne et ferrocene.

7. Biocapteur selon la revendication 1 , dans lequel le
systeme d'electrodes principales (19) et le systeme
de sous-electrodes (20) sont prevus sur des surfa-
ces differentes du substrat (61 ) en etant ecarte Tun
de I'autre, et la couche reactionnelle (50; 51; 52) est
fournie sur le systeme d'electrodes principales (1 9).

8. Biocapteur selon la revendication 1, dans lequel la
contre-electrode (7) du systeme d'electrodes prin-
cipales (19) est commune a la contre-electrode (9)
du systeme de sous-electrodes (20).

9. Biocapteur selon la revendication 1 , dans lequel le
systeme d'electrodes principales (19) est realise
avec un materiau comprenant du carbone comme
constituant principal.

10. Biocapteur selon la revendication 1, dans lequel le
sous-systeme d'electrodes (20) est realise avec un
materiau comprenant du carbone comme consti-
tuant principal.

11. Biocapteur comportant :

un substrat d'isolation electrique (1 ),
un systeme d'electrodes principales (19) forme
sur le substrat (1 ) et ayant une electrode de tra-
vail (6) et une contre-electrode (7),
une couche reactionnelle (5; 50; 51 ; 52) prevue
sur le systeme d'electrodes principales (19) et
contenant une oxydoreductase, et des accep-
teurs d'electrons; et

un systeme de sous-eiectrodes (20) comme re-
ference fourni avec un intervalle par rapport au
systeme d'electrodes principales (19) et a la
couche reactionnelle (5; 50; 51 ; 52) et ayant
une electrode de travail (8) et une contre-eiec-
trode (9), .

dans lequel un substrat contenu dans un liqui-
de echantillon est quantifie en reduisant les accep-
teurs d'electrons par des electrons produrts dans
une reaction du substrat contenu dans le liquide
echantillon et I'oxydoreductase, puis en mesurant

EP 0 537 761 B1

electrochimiquement ta quantite des accepteurs
d'electrons reduits.

12. Procede pour quantifier un substrat contenu dans

un liquide echantillon en utilisant un biocapteur, 5

le biocapteur comprenant un substrat desola-
tion electrique (1), un systeme d'electrodes
principales (19) forme sur le substrat (1) et
comportant une electrode de travail (6) et une 10
contre-electrode (7), une couche reactionnelle
(5; 50; 51 ; 52) prevue en contact avec ou dans
le voisinage du systeme d'electrodes principa-
les (1 9) et contenant une oxydoreductase, et
un systeme de sous-electrodes (20) comme re- '5
fSrence fourni sur le substrat (1) avec un inter-
vals par rapport au systeme d'electrodes prin-
cipales (1 9) et comportant une electrode de tra-
vail (8) et une contre-6lectrode (9), et
le procede comprenant les Stapes consistant a 2 o
detecter la presence du liquide echantillon sur
les deux systemes d'electrodes (19, 20) en 66-
tectant le changement de la caracteristique
Electrique entre le systeme d'electrodes princi-
pales (19) et le systeme de sous-Elect rodes 25
(20) et en appliquant ensuite une tension au
systeme d'electrodes principales (19) et au
systeme de sous-electrodes (20), respective-
ment.

13. Precede" pour quantifier un substrat contenu dans
un liquide echantillon en utilisant un biocapteur,

le biocapteur comprenant un substrat d' isola-
tion electrique (1), un systeme d'electrodes 35
principales (19) forme sur le substrat (1) et
comportant une electrode de travail (6) et une
contre -electrode (7), une couche reactionnelle
(5; 50; 51 ; 52) fournie en contact avec ou dans
le voisinage du systeme d'electrodes principa- 40
les (19) et contenant une oxydoreductase, et
un systeme de sous-electrodes (20) comme re-
ference fourni sur le substrat (1 ) avec un inter-
vals par rapport au systeme d'electrodes prin-
cipales (19) et ayant une electrode de travail 45
(8) et une contre-electrode (9), et
le precede comprenant les etapes consistant a
detecter le changement de la caracteristique
electrique entre I'electrode de travail (6; 8) et la
contre-electrode (7; 9) dans le systeme d'elec- so
trodes principales (1 9) et le systeme de sous-
electrodes (20), respectivement, et a determi-
ner la nature du liquide echantillon sur la base
de la difference de temps nEcessaire pour de-
tecter la caracteristique electrique dans les 5$
systemes d'electrodes principales et de sous-
electrodes.

EP 0 537 761 B1

fl
15 13 I

17 14

EP 0 537 761 B1

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